Next-generation sequencing (NGS) platforms are fundamentally altering genetic and genomic research by providing massive amounts of data in a low-cost, high-throughput format. The main drawback of existing technologies is the short sequence read lengths they produce. Existing library prep methods are also constrained in producing short inserts of only a few kb. As a result, de novo assembly of genomes is not practical with short read NGS technologies alone. Even with a high quality reference human genome, resequencing and assembly of new human genomes is a significant challenge when analyzing complex genomic regions. Haplotyping across more than a few kb is not achieved without resorting to cloned DNA. New tools that bridge the gap between massively parallel short read sequencing technologies (<1,500 bases) and the need for large scaffolds > 20 kb to assemble a genome are clearly needed. The SBIR Phase I grant proposal New Tools for Structural Variation Analysis, De Novo Assembly and Closing of Complex Genomes proposes to develop a new front end to NGS and the software to support it. The technology to construct clone-free 20-40 kb mate pair libraries from large randomly sheared DNA fragments does not yet exist. This technology will enable the accurate assembly of complex genomes, much like fosmid and BAC end sequences in conventional clone based strategies. The development of these tools could reduce manual closing costs and computational costs of genome assembly by orders of magnitude produce more complete and accurate genomes, enable the de novo sequencing of daunting genomes, and make personal genome resequencing and metagenomics tractable.